Towards detection of SARS-CoV-2 RNA in human saliva: A paper-based cell-free toehold switch biosensor with a visual bioluminescent output

Illustration of the assembly, distribution, and point-of-care use of a rapidly-deployable, cell-free COVID-19 biosensor: 1) Assemble: Assembling CFPS reagents by mixing E. coli lysate, murine RNase Inhibitor (mRI), energy sources, cofactors, and toehold switch riboregulator plasmid. 2) Print: aliquoting CFPS reagents onto paper substrates housed in a plastic test cassette. 3) Dehydrate: lyophilizing CFPS reagents on paper substrates. 4) Distribute. 5) Saliva sample: applying saliva samples onto cassette without pretreatments. 6) Reaction: bioluminescent protein expression in presence of target RNA (+), or ribosome detachment in absence of target RNA (-). 7) Visual result: bioluminescent output in the presence of target RNA and NanoLuc luciferase expression.